This invention was made with Government support awarded by the National Institute of Health. The government has certain rights in the invention. This invention relates to methods for isolating novel proteins. This invention also relates to cancer diagnostics and therapeutics.
In most eukaryotic cells, the cell cycle is governed by controls exerted during G1 and G2. During G2, cells decide whether to enter M in response to relatively uncharacterized intracellular signals, such as those that indicate completion of DNA synthesis (Nurse, Nature 344:503-508, 1990; Enoch and Nurse, Cell 65:921-923, 1991). During G1, cells either enter S or withdraw from the cell cycle and enter a nondividing state known as G0 (Pardee, Science 246:603-608, 1989). While the control mechanisms for these decisions are not yet well understood, their function is clearly central to processes of normal metazoa development and to carcinogenesis.
In yeast, and probably in all eukaryotes, the G1/S and G2/M transitions depend on a family of .about.34 kd protein kinases, the Cdc2 proteins, encoded by the cdc2.sup.+ (in S. pombe) and CDC28 (in S. cerevisiae) genes. Cdc2 family proteins from mammalian cells have been also identified. Some including Cdc2 (Lee and Nurse, Nature 327:31-35, 1987), Cdk2 (Elledge and Spotswood, EMBO J. 10:2653-2659, 1991; Tsai et al., Nature 353:174-177, 1991), and Cdk3 (Meyerson et al., EMBO J. 11:2909-2917, 1992) can complement a cdc28.sup.- S. cerevisiae for growth.
The activity of the Cdc2 proteins at the G2/M transition point is regulated in two ways: positively, by association with regulatory proteins called cyclins, and negatively, by phosphorylation of a tyrosine near their ATP binding site. At least one of these regulatory mechanisms is operative during G1 (see FIG. 1A). At this time, Cdc2 protein activity is regulated by facultative association with different G1 specific cyclins. In S. cerevisiae at least five putative G1 cyclins have been identified in genetic screens, including the products of the CLN1, CLN2, CLN3, HSC26 and CLB5 genes (Cross, Mol. Cell. Biol 8:4675-4684, 1988; Nash et al., EMBO J. 7:4335-4346, 1988; Hadwiger et al., Proc. Nat. Acad. Sci. U.S.A. 86:6255-6259, 1989; and Ogas et al., Cell 66:1015-1026, 1991). The CLN1, CLN2, and CLN3 proteins (here called Cln1, Cln2, and Cln3) are each individually sufficient to permit a cell to make the G1 to S transition (Richardson et al., Cell 59:1127-1133, 1989), and at least one of them (Cln2) associates with Cdc28 in a complex that is active as a protein kinase (Wittenberg et al., Cell 62:225-237, 1990). Recently, putative G1 cyclins have been identified in mammalian cells: Cyclin C, Cyclin D (three forms), and Cyclin E (Koff et al., Cell 66:1217-1228, 1991; Xiong et al., Cell 65:691-699, 1991). Each of these three mammalian cyclins complement a yeast deficient in Cln1, Cln2, and Cln3, and each is expressed during G1.
In S. cerevisiae, the synthesis, and in some cases, the activity of the G1 cyclins is under the control of a network of genes that help to couple changes in the extracellular environment to G1 regulatory decisions (FIG. 1A). For example, the SWI4 and SWI6 gene products positively regulate CLN1 and CLN2 transcription and may also positively modulate the activity of Cln3 (Nasmyth and Dirick, Cell 66:995-1013, 1991), the FAR1 product negatively regulates both CLN2 transcription and the activity of its product (Chang and Herskowitz, Cell 63:999-1011, 1990), and the FUS3 product negatively regulates Cln3 activity (Elion et al., Cell 60:649-664, 1990).
Several lines of evidence suggest that mammalian G1 to S transitions may be regulated by similar mechanisms: regulatory molecules (Cdc2 kinases and cyclins) similar to those found in yeast are observed in mammalian G1, and like S. cerevisiae, mammalian cells arrest in G1 when deprived of nutrients and in response to certain negative regulatory signals, including contact with other cells or treatment with negative growth factors (e.g., TGF-.beta.) (FIG. 1B). However, several considerations suggest that the higher eukaryotic G1 regulatory machinery is likely to be more sophisticated than that of yeast. First, in mammalian cells there appear to be more proteins involved in the process. At least ten different Cdc2 family proteins and related protein kinases (see Meyerson et al., EMBO J. 11:2909-2917, 1992) and at least three distinct classes of putative G1 cyclins (Koff et al., Cell 66:1217-1228, 1991; Matsushime et al., Cell 65:701-713, 1991; Motokura et al., Nature 339:512-518, 1991; Xiong et al., Cell 65:691-699, 1991) have been identified. Second, unlike yeast, the proliferation of most mammalian cells depends on extracellular protein factors (in particular, positive growth regulatory proteins), deprivation of which leads to arrest in G1. Third, arrest of many cell types during G1 can progress to a state, G0, that may not strictly parallel any phase of the yeast cell cycle.
Because proteins involved in controlling normal cell division decisions in mammals (e.g., humans) are also very likely to play a key role in malignant cell growth, identification and isolation of such proteins facilitate the development of useful cancer diagnostics as well as anti-cancer therapeutics. We now describe (i) a novel system for the identification of proteins which, at some time during their existence, participate in a particular protein-protein interaction; (ii) the use of this system to identify interacting proteins which are key regulators of mammalian cell division; and (iii) one such interacting protein, termed Cdi1, a cell cycle control protein which provides a useful tool for cancer diagnosis and treatment.